1. Field of the Invention
The invention relates to a method and a system for screening of a to-be-analyzed candidate as a skin-whitening agent.
2. Description of the Related Art
For the characteristics that they produce opticallly transparent embryos and that embryo development is rapid, zebrafish, which is a tropical freshwater fish with a binominal name of Danio Rerio, is considered an important vertebrate model organism in scientific research, such as in the fields of molecular genetics and developmental biology.
Since melanin pigment is inherently present in the body surface of zebrafish, no complex preparation procedure is necessary to induce melanogenesis for experimental purposes. Furthermore, since zebrafish has the advantages of being low-cost, small-sized (thus taking less space) and easily-bred, of having a fully-sequenced genetic code, and of requiring little test dosage during experimentation, the use of zebrafish as an in vivo model for screening of a to-be-analyzed candidate as a skin-whitening agent has gained increasing attention.
In the article by T. Y. Choi et al. and entitled “Zebrafish as a new model for phenotype-based screening of melanogenic regulatory compounds”, Pigment Cell & Melanoma Research, 2007, 20:120-127, the content of which is incorporated herein by reference, a method for screening of melanogenic regulatory compounds is disclosed to include the following steps. (1) Synchronized embryos were collected and dispensed in 96-well plates containing 200 μL embryo medium. (2) Test compounds were dissolved in 0.1% dimethyl sulfoxide (DMSO), and were then added to the embryo medium at 9 hour post-fertilization for 63 hours of exposure time. (3) 0.2 mM of 1-phenyl-2-thiourea (PTU) was used to generate transparent zebrafish without interfering with the developmental process as a standard positive control. (4) For observation, embryos were dechorionated by forceps, anesthetized in tricaine methanesulfonate solution, mounted in 3% methyl cellulose on a depression slide, and photographed under the stereomicroscope MZ16 (Leica). (5) The effects on the pigmentation of zebrafish were scored arbitrarily by the naked eye as follows: none or mild, <10%; moderate, 10-49%; profound, >50%.
Although the conventional method demonstrated the use of zebrafish for the screening of melanogenic regulatory compounds, it fails to obtain objective, reliable, and statistically significant results due to reliance upon the naked eye as a basis for determination.